Molecular Biology Section, Division of Biological Sciences, Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, California, USA.

Abstract

Foxo transcription factors regulate cell cycle progression, cell survival and DNA-repair pathways. Here we demonstrate that deficiency in Foxo3 resulted in greater expansion of T cell populations after viral infection. This exaggerated expansion was not T cell intrinsic. Instead, it was caused by the enhanced capacity of Foxo3-deficient dendritic cells to sustain T cell viability by producing more interleukin 6. Stimulation of dendritic cells mediated by the coinhibitory molecule CTLA-4 induced nuclear localization of Foxo3, which in turn inhibited the production of interleukin 6 and tumor necrosis factor. Thus, Foxo3 acts to constrain the production of key inflammatory cytokines by dendritic cells and to control T cell survival.

Increased number and activation of DCs in Foxo3Kca mice (a) Splenocytes from Foxo3Kca and wild-type littermates (n = 10 mice per group) were stained for CD11c expression and the total DC number was calculated. (b) CD11c+ DCs from naïve Foxo3Kca or wild-type littermates (n = 5 mice per group) were stained with antibodies specific for the indicated markers and analyzed by flow cytometry. The accumulation of data is graphed under each histogram (c) Absolute numbers of CD11c+CD11b+CD8−; CD11c+CD11b−CD8+ and CD11c+B220+ DCs in the spleen of Foxo3Kca and wild-type littermates was determined (n = 5 mice per group). (d) Expression of B7-1 and B7-2 on indicated DC subsets from Foxo3Kca and wild-type littermates was measured by flow cytometry. (e) Total splenic DCs (CD11c+) were purified from wild-type or Foxo3Kca mice and 400,000 cells were cultured for 24 h. Concentrations of IL-6, TNF, MCP-1, IL-10, IFN-γ and IL-12p70 in culture supernatants were measured by CBA. Data are representative of three independent experiments (a–b) or two independent experiments with at least three mice per group (c–e). (* signifies p<0.01, ** signifies p<0.005, *** signifies p<0.001, unpaired two-tailed Student t-Test).

Increased immunogenicity of LCMV-infected Foxo3−deficient DCs (a) Expression of activation markers on wild-type and Foxo3Kca DCs on day 3 post LCMV infection was analyzed by flow cytometry. (b) DCs isolated from day 3 LCMV infected Foxo3Kca mice or wild-type littermates were used to stimulate CFSE-labeled wildtype P14 CD8 T cells in the presence or absence of gp33 peptide. Bystander proliferation was assessed using CFSE-labeled wildtype OTI CD8 T cells stimulated in the presence or absence of OVA peptide. T cell proliferation was quantified by CFSE dilution and T cell viability was measured by 7-AAD and Annexin V staining after 3 days of culture. Numbers in dot plots indicate percentage of live CD8 T cells. (c) DCs purified from the spleen of uninfected mice were cultured and analyzed as described in (b), and cultured with T cells from P14 mice. Data are representative of two independent experiments with at least three mice per group (a and c) or three independent experiments (b).

Enhanced T cell response induced by Foxo3Kca BMDCs (a) Wild-type OTII CD4 T cells or wild-type P14 CD8 T cells were stimulated at various ratios with wild-type or Foxo3Kca BMDCs in the presence of OVA323–239 or gp33 peptide, respectively. After 3 days of culture, T cell accumulation was measured by CFSE dilution. (b) 1 × 105 OTII or P14 T cells were cultured with 3.3 × 102 wild-type or Foxo3Kca BMDCs in the presence or absence of the appropriate peptide. T cell death was assessed by 7-AAD staining. Numbers in dot plots indicate the percentage of 7-AAD+ CD8+ or CD4+ T cells. (c) OTII or P14 T cells were cultured with BMDCs for 3 days in the presence or absence of the appropriate peptide as in (b) and stained with 7-AAD and Annexin V. Numbers in dot plots indicate the percentage of viable CD4 or CD8 T cells. (d) OTII or P14 T cells were cultured as in (b) and the expression of Bcl-2 and Bcl-xL in total T cells or gated CD44lo T cells was analyzed by flow cytometry. The dashed line indicates the isotype control. (e) CD4 T cells purified from wild-type CD45.2 OTII mice were labeled with CFSE and injected intravenously into CD45.1 recipient mice. OVA323–239 (OVAp) pulsed or unpulsed BMDC from wild-type littermates or Foxo3Kca mice were then injected subcutaneously into the footpad of the same recipient mice. Three days later, CD4 CD45.1 cells in the draining LNs were enumerated and proliferation analyzed by CFSE dilution. Graphs show mean ± sem. n = 4 mice per group). Data are representative of at least six independent experiments (a–b), two independent experiments (c–d), or three independent experiments (e).

IL-6 synthesis by Foxo3-deficient DCs is involved in enhanced T cell survival. (a) Plasma from wild-type or Foxo3Kca mice (n = 4 mice per group) was collected on the indicated days post LCMV infection. The amount of IL-6 in the plasma was measured by CBA. (b) DCs (CD11c+) were purified from wild-type or Foxo3Kca uninfected mice or 3 days post LCMV infection (n = 4 mice per group). 200,000 cells were cultured for 24 h, and secreted IL-6 was measured using CBA. The relative expression of Il6 mRNA was measured by rtPCR (c) Wild-type OTII or P14 T cells were cultured in the presence of OVA323–339 or gp33 peptide-loaded wild-type or Foxo3Kca BMDCs as described in . IL-6 concentrations in the supernatants were assessed by ELISA. (d) BMDC, OTII and P14 T cells were purified after 2 days of culture as described in (c). Il6 mRNA was measured by RT-PCR and was normalized to Gapdh mRNA. (e,f) BMDCs generated from wild-type or Foxo3Kca mice were pulsed with OVA323–339 or gp33 peptide and used to activate wild-type OTII or P14 T cells, respectively, in the presence of an IgG1 isotype control antibody or IL6-specific blocking antibody (10 µg/ml). After 3 days of culture, T cell accumulation was measured by CFSE dilution and T cell death was measured by 7-AAD staining. (g) BMDC generated from wild-type mice were used to stimulate P14 T cells in the presence of increasing amounts of recombinant IL-6. After 3 days of culture, T cell death was assessed by 7-AAD staining. (h) Wild-type littermate or Foxo3Kca mice (n = 4 mice per group) were treated on day −1 and day 4 with 100 µg of anti IL-6Rα and infected at day 0 with LCMV Armstrong. The LCMV-specific T cell response was analyzed on day 8 post-infection by enumeration of CD8 and CD4 T cells producing IFN-γ after restimulation with gp33 and gp61, respectively. Data are representative of three independent experiments (a–f) or two independent experiments (g–h).

IL-6 and TNF production by stimulated DCs is inhibited by CTLA-4-Ig stimulation in a Foxo3-dependent manner. (a) 2×105 DCs purified from the spleen of wild-type littermate or Foxo3Kca mice were stimulated for 18 h with or without loxoribine along with increasing amounts of CTLA-4-Ig (µg/ml). IL-6 and TNF concentrations in the supernatants were assessed by immunoassay. DC viability was determined by 7-AAD staining (b) Foxo3 localization (green) was assessed by immunoflorescence after 18 h of stimulation with CTLA-4-Ig and/or loxoribine. Dapi, blue. (c) 1 × 105 OTII CD4 T cells or P14 CD8 T cells were cultured with 3.3 × 102 wild-type or Foxo3Kca BMDCs in the presence or absence of the appropriate peptide. 50 µg/ml of anti-CTLA-4 (9D9) was added to the culture. After 3 days, T cell accumulation was measured by CFSE dilution, and T cell death was assessed by 7-AAD and Annexin V staining. Numbers in dot plots indicate percentages of live CD4 or CD8 T cells. Data are representative of at least two independent experiments.